Prion protein (PrP) underlies a spectrum of diseases with no established treatment and devastating human and economic consequences. The "protein-only" hypothesis postulates that an abnormal prion protein conformation (PrPSc) propagates itself in an autocatalytic manner by recruiting normal isoform of the same protein (PrPC) and, therefore, acts as a transmissible agent of disease. The reconstitution of PrPSc in vitro from synthetic components has been difficult to achieve despite many years of effort. These difficulties are attributed to the lack of reliable biochemical markers of prion infectivity and to our poor understanding of the physical properties that are essential for infectivity. During the previous funding period, we developed the first experimental procedure for cell-free conversion of full-length PrP into self-propagating amyloid fibrils; we described several pathways of PrP polymerization; we introduced the most comprehensive mechanism of PrP conversion; we also established several novel assays including an immunoconformational assay for probing conformation within a single PrP fibril or particle. In the present application, we propose to elucidate PrPSc ultrastructure and to establish a link between infectivity and physical property of prion fibrils. The first specific aim will elucidate the substructure of PrPSc using novel immunoconformational assay developed in our laboratory combined with high resolution Atomic Force Microscopy. The second specific aim will elucidate the ultrastructure of prion fibrils generated in vitro, and the third specific aim is designed to test a relationship between conformational properties of the in vitro generated fibrils and their intrinsic infectivity. Such knowledge should lay the foundation for development of sensitive antemortem diagnostics and efficient therapeutics for treating prion diseases. [unreadable] [unreadable] [unreadable]